Method and apparatus for fractionation using generalized dielectrophoresis and field flow fractionation

ABSTRACT

The present disclosure is directed to a novel apparatus and novel methods for the separation, characterization, and manipulation of matter. In particular, the invention combines the use of frequency-dependent dielectric and conductive properties of particulate matter and solubilized matter with the properties of the suspending and transporting medium to discriminate and separate such matter. The apparatus includes a chamber having at least one electrode element and at least one inlet and one output port into which cells are introduced and removed from the chamber. Matter carried through the chamber in a fluid stream is then displaced within the fluid by a dielectrophoretic (DEP) force caused by the energized electrode. Following displacement within the fluid, matter travels through the chamber at velocities according to the velocity profile of the chamber. After the matter has transitted through the chamber, it exits at the opposite end of the chamber at a characteristic position. Methods according to the invention involve using the apparatus for discriminating and separating matter for research, diagnosis of a condition, and therapeutic purposes. Examples of such methods may include separation of mixtures of cells, such as cancer cells from normal cells, separation of parasitized erythrocytes from normal erythrocytes, separation of nucleic acids, and others.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularseparation and particle discrimination. More particularly, it concernsthe fractionation of particulate matter utilizing a combination ofelectrical, hydrodynamic or gravitational forces.

2. Description of the Related Art

The ability to identify, characterize and purify cell subpopulations isfundamental to numerous biological and medical applications, oftenforming the starting point for research protocols and the basis forcurrent and emerging clinical protocols. Cell separation has numerousapplications in medicine, biotechnology, and research in environmentalsettings. For example, cell separation can make possible life-savingprocedures such as autologous bone marrow transplantation for theremediation of advanced cancers where the removal of cancer-causingmetastatic cells from a patient's marrow is necessitated (Fischer,1993). In other applications, such as the study of signaling betweenblood cells (Stout, 1993), (Cantrell et al., 1992), highly purified cellsubpopulations permit studies that would otherwise be impossible.Current approaches to cell sorting most frequently exploit differencesin cell density (Boyum, 1974), specific immunologic targets (Smeland etal., 1992), or receptor-ligand interactions (Chess and Schlossman, 1976)to isolate particular cells.

These techniques are often inadequate and sorting devices capable ofidentifying and selectively manipulating cells through novel physicalproperties are therefore desirable. The application of the principles ofAC electrokinetics has been used for the dielectric characterization ofmammalian cells through the method of electrorotation (ROT) (Arnold andZimmermann, 1982; Fuhr, 1985; Holzel and Lamprecht, 1992; Wang et al.,1994) and for cell discrimination and sorting (Hagedorn et al., 1992;Huang et al., 1993; Gascoyne et al., 1992; Gascoyne et al., 1994; Huanget al., 1992). In these techniques, cells become electrically polarizedwhen they are subjected to an AC electric field. If that field isinhomogeneous, then the cells experience a lateral dielectrophoretic(DEP) force, the frequency response of which is a function of theirintrinsic electrical properties (Gascoyne et al., 1992). In turn, theseproperties depend strongly on cell composition and organization,features that reflect cell morphology and phenotype. Cells differing intheir electrical polarizabilities can thus experience differentialforces in the inhomogeneous electric field (Becker et al., 1994; Beckeret al., 1995). Analysis of the dielectrophoretic motion of mammaliancells as a function of applied frequency permits cell membranebiophysical parameters, such as capacitance and surface conductance, tobe probed. Because DEP effectively maps biophysical properties into atranslational force whose direction and magnitude reflects cellularproperties, some degree of separation occurs between particles ofdifferent characteristics. While DEP has been used on a microscopicscale to separate bacteria from erythrocytes (Markx et al., 1994),viable from nonviable yeast cells (Wang et al., 1993), anderythroleukemia cells from erythrocytes (Huang et al., 1992), thedifferences in the electrical polarizabilities of the cell types inthose various mixtures were greater than those to be expected in manytypical cell sorting applications.

Field flow fractionation (FFF) has also been generally employed forseparation of matter, utilizing particle density, size, volume,diffusivity, thickness, and surface charge as parameters (Giddings,1993). The technique can be used to separate many different types ofmatter, from a size of about 1 nm to more than about 100 micrometerswhich may include, for example, biological and non-biological matter.Separation according to field flow fractionation occurs by differentialretention in a stream of liquid flowing through a thin channel. The FFFtechnique combines elements of chromatography, electrophoresis, andultracentrifugation, and generally FFF requires the presence of a fieldor gradient to develop a differential flow. This differential flowcreates a flow profile which may be, for example, linear or parabolic. Afield is then applied at right angles to the flow and serves to drivethe matter into different displacements within the flow profile whichtravel at differing velocities. Fields may be based on sedimentation,crossflow, temperature gradient, centrifugal forces, and the like. Thetechnique suffers, however, from producing insufficiently pure cellpopulations, being too slow, or being too limited in the spectrum oftarget cells or other matter.

Thus, there exists a need in the art for highly discriminate separationof particulate matter, especially biological matter, that operateswithout physically modifying the structure of the matter to beseparated. Moreover, such an approach should allow for the sensitivemanipulation of such particles, which may include characterization andpurification of desired matter from extraneous or undesired matter.

SUMMARY OF THE INVENTION

The present invention seeks to overcome drawbacks inherent in the priorart by combining the use of frequency-dependent dielectric andconductive properties of particles with the properties of the suspendingand transporting medium. As used herein, the term "matter" is intendedto include particulate matter, solubilized matter or any combinationthereof. The invention provides a novel apparatus and novel methods bywhich different particulate matter and solubilized matter may beidentified and selectively manipulated. These particles may also becollected by changing the DEP force or the fluid flow characteristics.Utilizing the invention in this manner, particulate matter andsolubilized matter may be discriminated and separated. The apparatus andmethods of the present invention may discriminate different types ofmatter simultaneously.

The present invention provides a method and apparatus for thediscrimination of particulate matter and solubilized matter of differenttypes. This discrimination may include, for example, separation,characterization, differentiation and manipulation of the particulatematter. According to the present invention, the particulate matter maybe placed in liquid suspension before input into the apparatus. Thediscrimination occurs in the apparatus, which may be a thin, enclosedchamber. Particles may be distinguished, for example, by differences intheir density, size, dielectric permittivity, electrical conductivity,surface charge, and/or surface configuration.

The methods according to the present invention may be used todiscriminate particulate matter, including inorganic matter, such asminerals, crystals, colloidal, conductive, semiconductive or insulatingparticles and gas bubbles. The methods of the present invention may alsobe used to discriminate biological matter, such as cells, cellaggregates, cell organelles, nucleic acids, bacterium, protozoans, orviruses. Further, the particulate matter may be, for example, a mixtureof cell types, such as fetal nucleated red blood cells in a mixture ofmaternal blood, cancer cells such as breast cancer cells in a mixturewith normal cells, or red blood cells infested with malarial parasites.Additionally, the methods of the present invention may be used todiscriminate solubilized matter such as a molecule, or molecularaggregate, for example, proteins, or nucleic acids.

Particles to be discriminated may be any size. However, the presentinvention is generally practical for particles between ˜10 nm and ˜1 mm,and may include, for example, chemical or biological molecules(including proteins, DNA and RNA), assemblages of molecules, viruses,plasmids, bacteria, cells or cell aggregates, protozoans, embryos orother small organisms, as well as non-biological molecules, assemblagesthereof, minerals, crystals, colloidal, conductive, semiconductive orinsulating particles and gas bubbles. For biological applications usingliving cells, the present invention allows cells to be separated withoutthe need to alter them with ligands, stains, antibodies or other means.Cells remain undamaged, unaltered and viable during and followingseparation. Non-biological applications similarly require no suchalteration. It is recognized however, that the apparatus and methodsaccording to the present invention are equally suitable for separatingsuch biological matter even if they have been so altered.

The apparatus may include, for example, a chamber. The chamber may haveat least one inlet and one outlet port, an interior surface and anexterior surface. The chamber may further be designed to have structuralcharacteristics which cause a fluid or gas travelling through thechamber to travel at differing velocities according to a velocityprofile. The chamber may be rectangular in shape and may include, forexample, a top wall, bottom wall and two side walls. In certainembodiments, the chamber may be constructed so that the top wall andbottom wall are of a much greater magnitude than the side walls, therebycreating a thin chamber capable of creating a velocity profile. In otherembodiments, the chamber may be constructed so that the top wall andbottom wall are of a much smaller magnitude than the side walls, againcreating a thin chamber capable of creating a velocity profile.Alternately, the chamber may be of circular construction, triangular,rectangular, hexadecagonal, or of other geometrical shapes. As such, thepresent invention is not intended to be limited to a particulargeometric shape. The chamber according to the present invention may beconstructed of many different materials, for example, glass, polymericmaterial, plastics, quartz, coated metal, or the like.

The chamber includes at least one electrode element adapted along aportion or all of the chamber. Each of these one or more electrodeelements may be electrically connected to an electrical conductor. Inthe discussion which follows, the terms "electrode element" or"electrodes" will be used. As used herein, "electrode element" is astructure of highly electrically-conductive material over which anapplied electrical signal voltage is constant. The electrode elementsmay be any appropriate geometric shape such as, for example,castellated, rectangular, and the like. It is to be understood thatthese terms include all of the below described electrode configurations.An electrical signal generator, which may be capable of varying voltage,frequency, phase or any combination thereof, may transmit at least oneelectrical signal to the electrode elements. The electrode elements ofthe present invention may include, for example, a plurality of electrodeelements which may be connected to a plurality of electrical conductors,which in turn is connected to the electric signal generator.

The chamber according to the present invention may include a pluralityof electrode elements which comprise an electrode array. As used herein,an "electrode array" is a collection of more than one electrode elementin which each individual element may be displaced in a well-definedgeometrical relationship with respect to one another. This array may be,for example, a parallel array, interdigitated castellated array, apolynomial array, plane electrode, or the like. Further, the array maybe comprised of microelectrodes of a given size and shape, such as aninterdigitated array. The electrode array may be adapted along anyinterior or exterior surface of the chamber. Alternately, it isenvisioned that the electrode array may be incorporated into thematerial which comprises the chamber walls. In certain embodiments, theelectrode array may be a multilayer array in which conducting layers maybe interspersed between insulating layers. Fabrication of such anelectrode array is known in the art, and is similar to the fabricationof multilayer printed circuit boards. Further, the present invention mayhave a plurality of electrode arrays which may be adapted, for example,on opposing surfaces of the chamber. However. it may be possible toplace the plurality of electrode arrays on adjacent surfaces or on allsurfaces of the chamber.

The electrode elements may be adapted to be substantially longitudinallyor latitudinally along a portion of the chamber. Other configurations ofelectrode elements are contemplated by the present invention, such aselectrode elements adapted at angles to the chamber. It is also possibleto use a three-dimensional electrode element that may or may not beattached to the surface of the chamber. For example, electrode elementsmay be fabricated from silicon wafers, as is known in the art. If theelectrodes are adapted along the exterior surface of the chamber, it isenvisioned that a means of transmitting energy into the chamber, such asa microwave transmitter may be present. The electrode elements may beconfigured to be on a plane substantially normal or parallel to a flowof fluid travelling through said chamber, however, it is to beunderstood that the electrode elements may be configured at manydifferent planes and angles to achieve the benefits of the presentinvention.

When the electrode elements are energized by at least one electricalsignal from the electrical signal generator, the electrode elementsthereby create a spatially inhomogeneous alternating electric field,which may cause a DEP force on the particulate matter and solubilizedmatter having components normal to the fluid travelling through thechamber. This DEP force may be a conventional DEP force (cDEP), whichmay act in different directions with respect to the fluid, depending onthe configuration of the electrode elements. The cDEP force typicallyacts in a direction substantially normal to the electrode element plane,that is, the cDEP force typically forces matter towards or away fromthis plane. In certain embodiments, the DEP force may act solely in adirection normal to the fluid. As used herein, "a direction normal tothe fluid" means in a direction which is substantially non-opposing andsubstantially non-linear to the flow of a fluid traveling through thechamber. This direction may be for example, vertically, sideways, or inanother non-opposing direction. By effect of this DEP force, theparticulate matter and solubilized matter may be displaced to positionswithin the fluid. This displacement may be relative to the electrodeelements, or may relate to other references, such as the chamber walls.

The ratio of electrode element width to electrode element spacing may bemodified to change the particulate matter and solubilized matterlevitation height. That is, as used herein, "levitate" or "levitationheight" means that matter is displaced at different levels with respectto the electrode elements, in any direction. Specifically, by changingthis ratio, the electric field which is created is thereby altered. Whenthe electric field is thereby altered, in magnitude and/orinhomogeneity, the levitation height of the matter similarly changes.This levitation need not be in a vertical direction, and may includedisplacement in a horizontal direction, for example.

In the present invention, the cDEP force is dependent on the magnitudeof the spatial inhomogeneity of the electric field and the in-phase(real) part of the electrical polarization induced in matter by thefield. It is to be understood that the term "electrical polarization" isrelated to the well-known Clausius-Mossotti factor, described below.This field-induced electrical polarization is dependent on thedifferences between the dielectric properties between the matter and thesuspending medium. These dielectric properties in general arerepresented by dielectric permittivity and electrical conductivity. Thecombination of these two properties is known as complex permittivity.The cDEP force causes the matter to move towards or away from regions ofhigh electrical field strength, which in an exemplary embodiment, may betowards or away from the electrode plane.

The equation for the time-averaged conventional dielectrophoretic forcein an electric field strength having an rms value of E_(rms) is:

    F(t)=2πε.sub.m r.sup.3 Re f.sub.cm !▾E.sub.rms.sup.2                          ( 1)

where the factor f_(cm) is the well-known Clausius-Mossotti factordefined as f_(cm) (ε_(p) ^(*),ε_(m) ^(*))=(ε_(p) ^(*) -ε_(m)^(*))/(ε_(p) ^(*) +2ε_(m) ^(*)), and where ε_(p) ^(*) and ε_(m) ^(*) arethe complex permittivities of the matter and its suspending medium,respectively. The Clausius-Mossotti factor may also include other termsto account for other forms of electrical polarization or conductivityinduced or modified by the applied field, for example, the surfacecharge of the matter. In the force equation, r is the radius of thematter desired to be discriminated, Re f_(cm) ! is the real part(in-phase component) of the factor f_(cm), and ▾E_(rms) ² is themagnitude non-uniformity factor of the applied electric field. As seenfrom equation (1), if the in-phase part of the Clausius-Mossotti factoris oreater than zero, then the matter tends to move towards the strongfield. If the in-phase part of the Clausius-Mossotti factor is less thanzero, the matter tends to move towards the weak field.

In an alternate embodiment of the present invention, the electrode arraymay include at least three sets of electrode elements. As used herein,"set" is a group of individual electrode elements having a definedgeometric relationship to each other. Such a defined geometricalrelationship may include, for example, a triangle, circle, square, orcomplex shapes such as conic sections, and the like. It is to beunderstood that sets of electrode elements may be designed based onprinciples of electromagnetic theory. At least one applied electricalsignal having different phases may be applied to the at least three setsof electrode elements. For example, one applied signal may be adequatelydelayed by a capacitor or other time delay circuitry to provide each ofthe at least three sets of electrode elements a signal having adifferent phase. Alternatively, different signals having different phaserelationships may be provided to the electrode elements. These signalsthus create an electric field distribution that may be spatiallyinhomogeneous with respect to magnitude, and may travel through space byvirtue of the phase distribution.

It is noted that by applying at least one electrical signal havingdifferent phases to the at least three sets of electrode elements, atraveling wave dielectrophoretic (twDEP) force is created in addition tothe cDEP force. The twDEP force is dependent upon the phase distributionof the applied electric field (reflecting its movement through space,which is in the direction of small phase regions), and the out-of-phase(imaginary) part of the electrical polarization induced in the matter bythe field. The twDEP force causes the matter to move towards or awayfrom the direction of increasing phase values. The twDEP force typicallyacts in a direction substantially parallel to the electrode elementplane. In the case of twDEP, the equation for the time-averagedtravelling wave dielectrophoretic force is:

    F(t)=2πε.sub.m r.sup.3 Im(f.sub.cm)(E.sup.2.sub.x0 ▾Φ.sub.x +E.sup.2.sub.y0 ▾Φ.sub.y +E.sup.2.sub.z0 ▾Φ.sub.z)              (2)

where Im(f_(cm)) is the imaginary part (out-of-phase component) of thefactor f_(cm), and E² ▾Φ is the phase non-uniformity factor (whereE_(x0), E_(y0) and E_(z0) are the magnitudes of each electric fieldcomponent in the Cartesian co-ordinate frame, and Φ_(x), Φ_(y), andΦ_(z), are the phases of each field component). As seen from Equation(2), if the out-of-phase part of the Clausius-Mossotti factor is greaterthan zero, the force directs matter towards regions where the phases ofthe field components are larger. If the out-of-phase part of theClausius-Mossotti factor is less than zero, the force directs mattertowards regions where the phases of the field components are smaller.

The combination of the cDEP and twDEP forces is referred herein asgeneralized dielectrophoresis (gDEP). The combination of equations (1)and (2) therefore results in the equation for the time-averagedgeneralized dielectrophoretic force:

    F(t)=2πε.sub.m r.sub.3 (Re f.sub.cm !▾E.sub.rms.sup.2 +Im(f.sub.cm)(E.sup.2.sub.x0 ▾Φ.sub.x +E.sup.2.sub.y0 ▾Φ.sub.y +E.sup.2.sub.z0 ▾Φ.sub.z))             (3)

Thus, matter under the influence of one or both of these forces may bedisplaced to different positions within the fluid flowing in thechamber. It is noted that the cDEP force may be approximately four timesgreater than the twDEP force caused by the same electrical fieldstrength. It is to be understood that cDEP and twDEP are principalcomponents of gDEP, and other gDEP forces such as a combination of cDEPand twDEP, may also act on matter in an apparatus according to thepresent invention.

In an embodiment of the present invention which utilizes twDEP forces inaddition to cDEP forces, matter may be displaced in two-dimensions (timeand horizontal). As discussed below the cDEP force combined with fieldflow fractionation causes the matter to be displaced to differentpositions within the fluid flow. In addition, in an embodiment in whichthe electrode elements are configured substantially parallel to thefluid flow through the chamber, the twDEP force on matter actssubstantially parallel to the plane of the electrode elements, andsubstantially transverse to the fluid flow. Therefore, matter isdeflected laterally across the chamber from the original narrow flowstream in which it entered the chamber. The direction and magnitude ofthis deflection is a measure of the imaginary part of the dielectricproperties of the matter, namely the electrical polarization induced inthe matter by the field. The deflected matter therefore travels throughthe chamber and exits at positions that are laterally displaced from theport through which it entered.

Thus, the combined influence of the cDEP and twDEP forces results in atime and horizontal displacement of matter. Each component of thedisplacement represents different characteristics of the introducedmatter, and matter having different characteristics may experiencedifferent components of force in each direction. Therefore, matterdiscrimination may be achieved according to the time of travel throughthe chamber depending on the in-phase (real) part of the polarization ofthe matter, its density, surface configuration. volume, and thedielectric properties of the fluid. Matter discrimination according tothe lateral displacement of the matter from its inlet position dependson its volume and the out-of-phase (imaginary) part of its electricalpolarization, and the dielectric properties of the fluid. The outletport may be constructed so that matter having different lateralpositions at one displacement level may be separately discriminated. Forexample, it may be possible to utilize a laser as a tool to determinecharacteristics of matter exiting at selected lateral positions.

Common electrical conductors may be used to connect the one or more setsof electrode elements to the signal generator. The common electricalconductors may be fabricated by the same process as the electrodes, ormay be one or more conducting assemblies, such as a ribbon conductor,metallized ribbon or metallized plastic. A microwave assembly may alsobe used to transmit signals to the electrode elements from the signalgenerator. All of the electrode elements may be connected so as toreceive the same signal from the generator. It is envisioned that such aconfiguration may require presence of a ground plane. More typically,alternating electrodes along an array may be connected so as to receivedifferent signals from the generator. The electrical generator may becapable of generating signals of varying voltage, frequency and phaseand may be, for example, a function generator, such as a Hewlett Packardgenerator Model No. 8116A. Signals desired for the methods of thepresent invention are in the range of about 0 to about 15 volts, andabout 0.1 kHz to about 180 MHz, and more preferably between about 0 toabout 5 volts, and about 10 kHz to 10 MHz. These frequencies areexemplary only, as the frequency required for matter discrimination isdependent upon the conductivity of, for example, the cell suspensionmedium. Further, the desired frequency is dependent upon thecharacteristics of the matter to be discriminated. The discriminationobtained depends on the shape size and configuration of the electrodeelements, for example. In an exemplary embodiment, the signals aresinusoidal, however it is possible to use signals of any periodic oraperiodic waveform. The electrical signals may be developed in one ormore electrical signal generators which may be capable of varyingvoltage, frequency and phase.

A chamber according to the present invention may have at least one inletand outlet port. These ports may be the same port, or the chamber may beconstructed to have different ports. The outlet port may be arranged tobe vertically lower than the at least one inlet port. Such anarrangement thereby permits sedimentation of the particulate matter andsolubilized matter as it travels throughout the chamber. In addition tothe at least one inlet port and one outlet port, the chamber may alsoinclude one or more input ducts which allow the fluid to flow throughthe apparatus.

The outlet port of the chamber according to the present invention maytake many forms. Specifically, the outlet port may be a single port, ora plurality of ports, or an array of ports. The outlet port, forexample, may be located along the entire width or a part of the width ofthe chamber. The outlet port may be adapted to receive matter of variousshapes and sizes. For example, the size of the outlet port may vary fromapproximately twice the size of the matter desired to be discriminatedto the entire width of the chamber. In one embodiment, the outlet portmay be constructed of one or more tubing elements, such as TEFLONtubing. The tubing elements may be combined to provide an outlet porthaving a cross section comprised of individual tubing elements. Further,for example, the outlet port may be connected to fraction collectors orcollection wells which are used to collect separated matter. As usedherein, "fraction collectors" and "collection wells" include storage andcollection devices for discretely retaining the discriminatedparticulate and solubilized matter. Other components that may beincluded in the apparatus of the present invention are, for example,measurement or diagnostic equipment, such as cytometers, lasers,particle counters and spectrometers.

After being displaced within the fluid travelling through the chamber ofthe present invention, the displaced matter may exit from the outletport or ports at a time proportionate to the displacement of the matterwithin the fluid. Specifically, matter at different levels ofdisplacement within the fluid travels at different speeds. Therefore,the displaced matter is discriminated by its displacement within thefluid flow. The position of the particulate matter and solubilizedmatter within the fluid causes the matter to travel through the chamberat velocities according to the velocity profile of the chamber.

This velocity profile may be, for example, a hydrodynamic fluid profilesuch as a parabolic flow profile. The velocity profile may be determinedby knowing the flow rate of the fluid, and the chamber width andthickness. The average velocity profile may then be calculated accordingto the equation:

    velocity profile=(flow rate)/(chamber width×chamber thickness).(4)

Parameters that determine the velocity profile of the fluid flow include(but are not limited to): the chamber width or thickness, which in arectangular embodiment may be the distance between opposing walls;constrictions or expansions of the fluid flow path which may include,for example, those arising for a non-parallel disposition of opposingchamber walls, or from the presence of suitably-placed obstructions orvanes; surface roughness of the chamber walls; structural features ofthe chamber walls that give rise to periodic or aperiodic modificationsof the thickness of the fluid stream, including the electrode elementsand other surface structural configurations, and the geometrical form ofthe chamber which may be, for example, rectangular, circular,wedge-shaped, stepped, or the like.

Embodiments of the present invention may have, for example, one inletport adapted to receive the particulate matter to be discriminated. Theinlet port may be located, for example, close to the top of one end ofthe chamber. This apparatus may also include one or more ducts tointroduce a fluid that travels through the chamber. The ducts, which maybe arranged substantially along the entire width of the input end of thechamber, serve to introduce a sheet of fluid that travels throughout thechamber in a substantially linear direction. As used herein, a "sheet"of fluid may be a flow of fluid or gas entering the chamber at asubstantially uniform flow rate. The introduced fluid carries theparticulate matter through the chamber. Following transit through thechamber, fluid leaves at the opposite end. This exit end of the chambermay include, for example, one or more exit ports, which may be arrangedin one or more arrays of exit ports.

Different electrical signals (frequency or magnitude or both) may beapplied to electrode elements located on each of the side walls. Thereis a synergistic interaction between these different electrical signalswhich creates an inhomogeneous electric field. Different matterequilibrates at different characteristic distances from the side wallsof the chamber based on this synergistic interaction of the differingelectrical signals, herein an "equilibrium position." The equilibriumposition is therefore caused by cDEP forces on the particles.Additionally, twDEP forces may impact this equilibrium position. Thisequilibration position depends on the dielectric and conductiveproperties of the matter, the magnitude and frequency of the electricalfields applied to the electrodes on the opposing chamber walls, fluiddensity, viscosity, and flow rate. The equilibrium position of matterdepends on the synergism of the different electrical signals actingwithin the chamber to levitate the matter. The velocity of the differentmatter within the fluid is controlled by the velocity profile of thefluid. This velocity profile has a maximum rate towards the center ofthe chamber, with this rate proportionately diminishing as distance fromthe side walls decreases. Because of this velocity profile, matter thathas equilibrated at different equilibrium distances from the chamberwalls will be carried at different velocities and therefore take varyingamounts of time to traverse the chamber.

The distance that matter sediments during its passage across the chamberwill depend upon its transit time, as gravitational forces act on thematter during its transit through the chamber, and is known as a"sedimentation effect." Consequently, different particles will sedimentto different dcpths based upon the transit time of matter through thechamber. Particle sedimentation also depends on matter characteristics,such as size, mass, and volume, for example. Therefore, the timerequired for particles to travel across the entire length of the chamberis controlled by the fluid flow profile. The placement of particleswithin the fluid flow profile is in turn determined by the synergism ofthe differing electrical signals. Particles with differentcharacteristics may exit the chamber through different outlet portswhich may be placed at different heights with respect to the inletports. Discrimination may be accomplished either in "batch mode" or in"continuous mode." In batch mode, an aliquot of particles is injectedand collected with respect to the time of transit for the particles andthe height of exit at the outlet ports. In continuous mode, a constantstream of particles is injected into the inlet port, and matter emergingat different heights are continuously collected.

A further embodiment of the chamber having two facing electrode arraysadapted on opposing surfaces is also possible. In this design, theelectrode arrays are arranged along the opposing surfaces so that theindividual electrode elements are substantially parallel to thedirection of fluid flow. However, it may be possible to create thedesired DEP forces by electrodes perpendicular or in other directions tothe flow of fluid. Different electrical signals (frequency, magnitude,and phase, or a combination thereof) may be applied to the facingelectrodes from the signal generator so that particles experiencedifferent cDEP and/or twDEP forces. The cDEP forces may act to displacematter to or away from the electrode plane, based on the magnitude ofthe field inhomogeneity. The twDEP forces may act in a planesubstantially parallel to the electrode arrays, based on the phaseinhomogeneity of the field produced by the electrode arrays.

The cDEP forces in combination with field flow fractionation causematter to be displaced characteristic distances with respect to theelectrode arrays. This characteristic distance is a function of thematter's dielectric and conductive properties, the magnitude andfrequency of the electrical fields applied to the electrodes on thefacing chamber walls, and the fluid density, viscosity and flow rate.Matter therefore equilibrates at an equilibrium position with respect tothe electrode arrays dependent on these characteristics. Because of thevelocity profile set up in the chamber, matter at different equilibriumpositions travels at different velocities and therefore takes differentamounts of time to travel through the chamber.

In a further embodiment, the electrode arrays may be arranged onopposing vertical walls of the chamber. In this embodiment, theelectrode arrays may be arranged at right angles to the flow of fluidthrough the chamber. In addition to displacement caused by the DEPforces, matter also sediments as it travels through the chamber and thedistance that the matter sediments depends upon the time required totraverse the chamber and the sedimentation rate of the matter. Thedistance of matter sedimentation is also affected by the twDEP force.Specifically, the twDEP force acts in a substantially vertical directionwhen the electrode arrays are located on the side walls of the chamber.Therefore the twDEP force component adds to or opposes the sedimentationforces acting on the matter. Thus the net vertical displacement whichthe matter experiences as it traverses the chamber is modified.

Matter discrimination in this embodiment may be accomplished in acontinuous mode, whereby a constant stream of matter is injected intothe at least one inlet port, and matter emerging at differing verticaldisplacements is continuously collected at the outlet ports.Alternately, it may be possible to operate this embodiment in a "pulse"mode. In pulse mode, a batch of matter may be injected into the at leastone inlet port of the chamber and electrical signals are applied for adefined amount of time. Then the signals are turned off, and the matteris allowed pass through the chamber under the influence of the fluidflow and be collected at the at least one outlet port. Then, anotherbatch may be injected for similar processing.

To further achieve the benefits of discrimination, the carrier fluidcharacteristics at different vertical displacements may be varied bymodifying for example, flow rate, fluid density, viscosity, dielectricpermittivity, pH and conductivity of the carrier fluid. In this way,additional matter characteristics may be exploited for particulardiscrimination applications.

The methods and apparatus of the present invention introduce for thefirst time the use of the frequency-dependent dielectric and conductiveproperties of particles as well as those of the suspending medium. Thesenew criteria for particle fractionation allow sensitive manipulation ofparticles because the dielectrophoretic force is large and stronglydependent on particle properties. Appropriate choices of the suspendingmedium and applied field conditions allow for high levels ofdiscrimination.

Previously reported field flow fractionation techniques have limitationsfor biological samples because of the narrow range of particledensities, demanding complex centrifuges and centrifugation techniquesfor good discrimination. The cDEP affinity method demands largedifferences in the dielectric characteristics of the particles to beseparated so that selected particulate matter and solubilized matter canbe completely immobilized while others are swept away by fluid flowforces. Since, for biological cells, damage can occur at high electricfield strengths, there is a practical limitation to the maximum cDEPforce that can be applied and this in turn limits the maximum fluid flowrate in the cDEP affinity approach. This may result in a slow cellsorting rate. In the methods of the present invention, these limitationsare substantially reduced. Furthermore, the cDEP affinity method of theprior art utilizes the dielectrophoretic force component that generallyimmobilizes particles on electrode elements. The cDEP/FFF approach ofthe present invention utilizes the cDEP component in a direction whichmay be normal to the flow.

Also, in the present invention, the flow profile is an active mechanismfor the separation and discrimination of particles, and thedielectrophoretic force (mainly the force component in the directionnormal to the fluid flow profile) is the primary means by which theheight of particles in the fluid stream is controlled. As discussedabove, the fluid profile may be controlled by apparatus design, fluidvelocity, density and the like. By combining FFF and dielectrophoreticforces, the present invention takes advantage of particle volume anddensity in synergism with the frequency-dependent particle dielectricand conductive properties as well as surface configuration. Theoperation of an apparatus according to the present invention may becontrolled by varying experimental conditions including, but not limitedto, the particle suspending medium conductivity and permittivity, thefluid flow rate, viscosity and density, the applied electrical fieldstrength and the applied frequency. This utilization of many parametersin setting the operational conditions for fractionation greatlyincreases the ability to discriminate between different particulatematter and solubilized matter. In the methods according to the presentinvention, particles emerging from the outlet ports of the apparatus maybe collected, for example, by one or more fraction collectors.Furthermore, when necessary or desired, particles may be transferred tocollection wells containing appropriate solutions or media, such asneutral salt buffers, tissue culture media, sucrose solutions, lysingbuffers, solvents, fixatives and the like.

In an illustrative embodiment, the chamber may be constructed in arectangular shape using, for example, two glass slides as chamber walls.These chamber walls may be spaced apart by spacers to create therectangular design. These spacers may be made of, for example, glass,polymeric material such as TEFLON, or any other suitable material. Thesize of the chamber and spacing between chamber walls is dependent onthe size of the particles which are to be discriminated. To practice themethods of the present invention, an apparatus may have spacing betweenabout 100 nm and about 1 mm, and more preferably between about 20microns and about 200 microns in an illustrative embodiment for thepurpose of discriminating mammalian cells. Further, a longer chamber maybe desired to permit greater discrimination throughput. An apparatusaccording to the present invention can discriminate cells at a ratebetween about 1000 and about 3 million cells per second. Factors thatdetermine discrimination rate include, for example, the dielectricproperties of the particles to be discriminated, the electrode design,length of the chamber, fluid flow rate, and frequency and voltage of theelectrical signals. The chamber dimensions may be chosen to beappropriate for the input matter type, characteristics, and degree ofdiscrimination desired or required.

In other embodiments, one or more surfaces of the chamber may support anelectrode array. The electrode array may be a microelectrode array of,for example, parallel electrode elements. In certain embodiments, theelectrode elements may be spaced about 20 microns apart. The apparatusmay accommodate electrode element widths of between about 0.1 micronsand about 1000 microns, and more preferably between about 1 micron andabout 100 microns for embodiments for the discrimination of cellularmatter. Further, electrode element spacing may be between about 0.1microns and about 1000 microns, and for cellular discrimination morepreferably between about 1 micron and about 100 microns. Alteration ofthe ratio of electrode width to electrode spacing in the parallelelectrode design changes the magnitude of the dielectrophoretic forceand thereby changes the particle levitation characteristics of thedesign. The electrode elements may be connected to a common electricalconductor, which may be a single electrode bus carrying an electricalsignal from the signal generator to the electrode elements. Alternately.electrical signals may be applied by more than one bus which providesthe same or different electrical signals. In certain embodiments,alternate electrode elements may be connected to different electrodebuses along the two opposite long edges of the electrode array. In thisconfiguration, alternate electrode elements are capable of deliveringsignals of different characteristics. As used herein, "alternateelectrode elements" may include every other element of an array, oranother such repeating selection of elements. The electrode elements maybe fabricated using standard microlithography techniques that are wellknown in the art. For example, the electrode array may be fabricated byion beam lithography, ion beam etching, laser ablation, printing, orelectrodeposition. The array may be comprised of for example, a 100 nmgold layer over a seed layer of 10 nm chromium.

An apparatus according to the present invention may be used with variousmethods of the present invention. For example, an apparatus according tothe present invention may be used in a method of discriminatingparticulate matter and solubilized matter utilizing dielectrophoresisand field flow fractionation. This method includes the following steps.First, a carrier medium, such as a cell suspension medium, tissueculture medium, a sucrose solution, or the like, which may include thematter to be discriminated, may be introduced into one or more inletports of the chamber. This introduction causes the carrier medium totravel through the chamber according to a velocity profile. At least onealternating electrical signal may be applied to the one or moreelectrode elements at different phases, which creates a travellingelectric field, which may also be spatially inhomogeneous, within thechamber. As used herein, "different phases" means that adjacentelectrode elements within an array may receive signals having differentphases. However, many electrode elements may receive signals of the samephase. For example, four adjacent electrode elements may receive signalsof 0°, 90°, 180° and 270° respective phase. The next four adjacentelectrode elements may receive signals having this same phaserelationship. Many other such phase relationships between electrodeelements may be used, and the sequence and number of electrode elementswhich have the same phase may be varied.

The field created by the different phases causes the matter within thechamber to be displaced to positions within the carrier medium. Thus,the matter is discriminated according to its positions within thecarrier medium. Specifically, the matter may be discriminated, forexample, according to the velocity profile of the carrier medium,because the velocity profile of the causes the matter at differentpositions within the chamber to travel at differing velocities. Tofurther discriminate matter, the electrical signal may be varied(frequency, magnitude, phase or any combination thereof). Such a changethereby causes a change in the travelling alternating electric fieldwhich, in turn, changes the displacement of the matter.

Another method according to the present invention includesdiscriminating particulate matter and solubilized matter utilizingdielectrophoresis and field flow fractionation according to thefollowing steps. First, the matter is introduced into at least one inletport of a chamber according to the present invention. Next, a transportfluid, which may be, for example, a tissue culture medium or a gas, isintroduced into at least one duct. The effect of this fluid in thechamber causes a fluid flow in the chamber at a speed according to thevelocity profile within the chamber. At least one electrical signal isapplied to electrode elements at different phases. These energizedelectrodes thereby create a travelling electric field, which may also bespatially inhomogeneous, within the chamber. The field causes a DEPforce on the matter causing the matter to be displaced to a positionwithin the transport fluid. As this transport fluid is subjected to avelocity profile, the matter is thereby partitioned according to itsposition within the transport fluid flow. It is further possible tocollect the matter at one or more outlet ports. Moreover, the matter maybe collected at a time dependent upon the velocity profile of thetransport fluid.

There are further steps possible to more precisely discriminate matter.These steps include the following. First, the alternating electricalsignal or signals may be selected at a frequency and voltage combinationwhich causes the matter to be either attracted towards or repelled fromthe electrode elements. By doing so, the matter is more clearlydisplaced within the transport fluid. By application of such a voltageand frequency combination. it is possible to hold the matter in closeproximity to the electrode elements.

It is possible to select a frequency to attract desired or nondesiredmatter. As used herein, desired matter may be any matter which isdesired to be discriminated and collected for further use. For example,the separation of normal blood cells from a sample including cancercells may be desired for use in returning these normal cells into apatient's bloodstream. Nondesired matter may be matter which is desiredto be discriminated for other purposes. For example, cancer cells from apatient's blood or bone marrow may be discriminated so that a sample ofblood not containing the cancer cells may be returned to the patient.

A method for discriminating such a combination of matter may include thefollowing. A frequency is selected so that the nondesired matter is heldin close proximity to the electrode elements and the desired matter ispartitioned from the nondesired matter by the fluid flow. This frequencymay be known as a holding frequency. The fluid flow then carries thedesired matter to the outlet port or ports of the chamber, where it maybe collected. After collection, the desired matter may, for example, bereturned to a patient's bloodstream or bones, or it may be used in adiagnostic manner. Then, to clear the chamber, the frequency may bechanged, or the voltage itself may be turned off. This will cause thenondesired matter to be released from close proximity to the electrodeelement and will be partitioned by the fluid flow. This nondesiredmatter may then flow through the chamber in the fluid, and may becollected, if desired. After collection, the nondesired matter may beused, for example, for diagnosis or other purposes.

In an alternate embodiment, it may be possible to hold desired matter inclose proximity to the electrode elements, and first partition thenondesired matter by the fluid flow, following the same steps outlinedabove.

The apparatus and methods of the present invention may be used for anumber of different useful manners. For example, the methods accordingto the present invention may be used to determine characteristics of anunknown particulate matter and unknown solubilized matter in a sample ofmatter. These characteristics can then be compared to known matter.Additionally, the methods of the present invention may be used todiagnose a condition by determining a presence of unidentifiedparticulate matter and unidentified solubilized matter in a patientsample. This unidentified matter may be, for example, the presence of acancer, a virus, parasite, or the like. After determining the presenceof a condition, the methods of the present invention may be used totreat the condition by using an apparatus according to the presentinvention to discriminate the cancer, virus, parasite or the like fromnormal blood or bone marrow cells.

"Manipulation" as used in relation to the present invention may include,for example, characterization, separation, fractionation, concentrationand/or isolation. Typical biological applications for the device usefulfor specific products and services include the manipulation of tumorcells, such as epithelial tumor cells or leukemia cells, from blood andhemopoietic stem cells, purging of tumor cells from bone marrow andhemopoietic stem cells and mixtures with other normal cells, purging ofresidual T-lymphocytes from stem cells, and enrichment of specifictarget cell types including tumor cells, stem cells, etc. Also includedis the manipulation of leukocyte cell subpopulations, removal andconcentration of parasitized erythrocytes from normal erythrocytes inmalaria and of other parasitized cells from their normal counterparts,manipulation of cells at different phases of the cell cycle,manipulation of viable and non-viable cells, manipulation of free cellnuclei, and manipulation of nucleated fetal erythrocytes from maternalblood for further analysis including genetic testing. Moreover, theinvention contemplates the manipulation of bacteria, viruses, plasmidsand other primitive organisms from water, blood, urine, cell mixturesand other suspensions, manipulation and identification of tumor cells inbiopsies, plaques and scrape tests including Pap smears, and themanipulation and identification of metastatic tumor cells from cellmixtures.

With different and smaller electrode geometries, it is contemplated thatthe technology can be used for molecular applications includingmanipulation of DNA or RNA molecules and/or DNA or RNA fragmentsaccording to their molecular weight, folding characteristics anddielectric properties, manipulation of chromosomes, manipulation ofspecific protein/DNA and protein/RNA aggregates, manipulation ofindividual proteins from a mixture, and manipulation of specificsubcellular molecular complexes and structures.

In order to optimize particle discrimination in different applicationsit is understood that the present invention may encompass use ofspecifically-targeted electrodes and chamber designs. These designsshould provide a sensitive dependency of the height of particlelevitation on the particle dielectric properties. For example,alteration of the ratio of electrode width to electrode spacing in theparallel electrode design changes the vertical component of thedielectrophoretic force and thereby changes the particle levitationcharacteristics of the design. Other strategies for providing improvedparticle discrimination include, for example, using more than two setsof electrode elements with different frequencies and/or voltages appliedto them and the exploitation of synergism between electrical signalsapplied to electrode arrays on both the chamber bottom and top walls. Inaddition, dielectric (i.e. non-conducting) elements can be placed withinthe chamber to modify both the electrical field distribution and thehydrodynamic flow profile. The electrode element size and shape may bedesigned to optimize discrimination. Furthermore, several electrodegeometries (energized with the same or different electrical signals) canbe connected serially so as to provide for stepwise discriminationbetween different particulate matter and solubilized matter. Differentchamber configurations can also be used in series. Finally, cells thathave been separated by an upstream cDEP/FFF or gDEP/FFF configurationcan be collected and held downstream by cDEP or gDEP trapping forcharacterization.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A is a block diagram of an apparatus according to the presentinvention.

FIG. 1B is a block diagram of an apparatus according to the presentinvention in which an electrode array is positioned normal to a fluidflow.

FIG. 1C is a block diagram of an apparatus according to the presentinvention in which an electrode array is positioned parallel to a fluidflow.

FIG. 2A is a side view of an apparatus according to the presentinvention which describes a typical trajectory of matter introduced intothe apparatus.

FIG. 2B is a top view of the apparatus of FIG. 2A which describes atypical trajectory of matter introduced into the apparatus.

FIG. 2C is an end view of the apparatus of FIG. 2A.

FIG. 3A is a graphical representation of HL-60 cells exiting anapparatus according to the present invention under the influence offield flow fractionation, as a function of time.

FIG. 3B is a graphical representation of HL-60 cells exiting anapparatus according to the present invention under the influence offield flow fractionation, as a function of time.

FIG. 3C is a graphical representation of HL-60/Human Whole Blood cellsexiting an apparatus according to the present invention under theinfluence of field flow fractionation, as a function of time.

FIG. 4A is a graphical representation of DS19 cell levitation heightunder the influence of cDEP as a function of frequency.

FIG. 4B is a graphical representation of DS 19 cell levitation heightunder the influence of cDEP as a function of voltage.

FIG. 5A is a graphical representation of velocity of HL-60 cellstravelling through an apparatus according to the present invention underthe influence of combined cDEP/FFF forces as a function of frequency.

FIG. 5B is a graphical representation of velocity of MDA 468 cellstravelling through an apparatus according to the present invention underthe influence of combined cDEP/FFF forces as a function of frequency.

FIG. 5C is a graphical representation of velocity of MDA 435 cellstravelling through an apparatus according to the present invention underthe influence of combined cDEP/FFF forces as a function of frequency.

FIG. 5D is a graphical representation of velocity of MDA 435 cellstravelling through an apparatus according to the present invention underthe influence of combined cDEP/FFF forces as a function of voltage.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following examples are included to demonstrate illustrativeembodiments of the invention. It should be appreciated by those of skillin the art that the apparatus and techniques disclosed in the exampleswhich follow represent devices and techniques discovered by the inventorto function well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention. While thefollowing examples use the term particle, the skilled artisan willrealize that the present apparatus and methods are suitable tosolubilized matter as well.

EXAMPLE I

FIG. 1A shows one exemplary embodiment of an apparatus according to thepresent invention. In this figure, the electrode array 5 is placed onthe bottom of a chamber 10; however it is contemplated that theelectrode array may be placed on the top and/or bottom walls and/or sidewalls of a chamber constructed in accordance with the present invention.As shown in FIG. 1B, the electrode array 5 may be placed along a chamberwall in a position normal to a flow of fluid 35 through the chamber 10.It is to be noted that the array may be adapted at any angle withrespect to the fluid flow, for example, parallel or at any other angle.In this embodiment, the walls are aligned to create a thin chamber. Thewalls are spaced apart by a spacer 20, which may be, for example,constructed of the same material as the chamber walls, or a TEFLONspacer, a sealing compound, or any other dielectric or conductivematerial. Electrical signals applied to the electrode array create aninhomogeneous alternating electric field that varies with the frequencyand magnitude of the input signal. In a particular embodiment, theelectrode element 5 may be adapted to be substantially normal to thefluid flow 35, as shown in FIG. 1B. Further electric conductors, whichmay be electrode buses 40 and 45 may provide electrical signals toalternate elements of electrode array 5. The strength of the electricfield is dependent on the applied voltage, position within the chamber,and the size and spacing of electrode elements. For manipulation ofmammalian cells, the field strength may be on the order of approximately1×10⁶ V/m, although this may be much higher for matter placed in an oilmedium. The particulate matter desired to be discriminated is introducedinto the chamber in a carrier medium that flows into at least one inletport 15. There may be more than one inlet port however, which permitsinput of the carrier medium. The carrier medium may be input by adigital syringe pump, a manual syringe, a peristaltic pump, a gravityfeed catheter, or the like. As discussed above, the particulate mattermay include, for example, biological molecules and non-biologicalmolecules. Also, the matter may include solubilized matter. The carriermedium may be, for example, an eluate consisting of a cell-freesuspension buffer, including a mixture of sucrose and dextrose, tissueculture medium, non-ionic or zwitter ionic solutes, or other suspensionmediums or non-biological oils, solvents such as phenol alcohol, CCl₄,ethylene glycol, or others known in the art. Alternately, one or moreducts 25 may be provided to input a fluid which may be flowed throughchamber 10.

The carrier medium is caused to flow through the chamber and therebycreate a laminar flow profile in which the fluid flow rate increaseswith increasing distance from the chamber top and bottom walls, andreaches its maximum at the center. However, by adjusting the shape ofthe chamber, for example, a flow profile may be created in which themaximum is at a location other than the center of the chamber. In anexemplary embodiment, this flow rate may be on the order of about 0.1μl/min. to about 100 μl/min., and more preferably about 1 μl/min. toabout 100 μl/min. The electric field applied to the electrode elements 5creates conventional dielectrophoretic forces on the particles inaccordance with their dielectric and conductive properties as well asthose of the carrier medium.

By controlling the frequency and/or intensity of the applied electricfield, the component of the dielectrophoretic force that is normal tothe direction of the carrier medium is controlled so as to cause theparticles to equilibrate at characteristic distances from the electrodeelement 5 creating the electric field. In particular embodiments, thedielectrophoretic force may act solely in a vertical direction so as tocause the particles to equilibrate at levitation heights above thechamber bottom wall. Such a force may be present in a vertical chamberor a chamber adapted for use in space. This dielectrophoretic forceoperates in conjunction with the action of the combined hydrodynamic andgravitational forces. Since the dielectrophoretic force acting on eachindividual particle depends upon its dielectric permittivity andelectrical conductivity at the applied frequency, as well as upon itsvolume, particles having different properties will be positioned atdifferent distances from the electrode element creating the electricfield. Because the fluid at different heights above the chamber bottomwall flows at different velocities, particles having differing physicalproperties will travel through the chamber 10 at different speeds andemerge at an outlet port 30 at different times. It is to be understoodthat there may be more than one outlet port from which to collect theparticulate matter which exits the chamber 10.

The chamber 10 of FIG. 1A may also utilize twDEP forces in addition tocDEP forces and field flow fractionation, thereby displacing matter intwo-dimensions (time and horizontal). In this embodiment, however, theelectrode array may be adapted to be substantially parallel to the fluidflow, as shown in FIG. 1C. As discussed above, the cDEP force on thematter causes it to be displaced to a position within the fluid flow. Inaddition, when the electrode elements 5 are energized with differentphases, a twDEP force on matter which acts substantially parallel to theplane of the electrode elements 5, and substantially transverse to thefluid-flow occurs. Therefore, matter is deflected laterally across thechamber 10 from the original narrow flow stream in which it entered thechamber 10. The deflected matter therefore travels through the chamber10 and exits through the outlet port 30 at positions laterally displacedfrom the inlet port 15 through with it entered. Thus, the combinedinfluence of the cDEP and twDEP forces in this embodiment results in atime and horizontal displacement of matter.

EXAMPLE 2

FIG. 2A shows a second embodiment of an apparatus according to thepresent invention that includes a chamber 10 having two facing electrodearrays 5, as shown in FIGS. 2B and 2C, on opposite surfaces of thechamber. The chamber is turned so that the electrode planes 5 standsubstantially vertical. In this embodiment, the chamber is arranged sothat the thin sides of the chamber are vertically arranged. It isunderstood, however, that the electrode planes need not be onlyvertical, and the present invention contemplates adapting the apparatusat varying angles. Different electrical signals (frequency andmagnitude) are applied to the facing electrodes from the signalgenerator so that particles experience different cDEP forces from thefield produced by each array 5. Further, within each facing electrodearray 5, different electrical signals may be provided by the signalgenerator to create an inhomogeneous alternating electric field.

This alternate apparatus may have, for example, one inlet port 15adapted to receive the particulate matter to be discriminated. The inletport 15 may be located, for example, close to the top of one end of thechamber 10. This apparatus may also include one or more ducts 25 tointroduce a fluid that travels through the chamber 10. The ducts 25,which may be arranged substantially along the entire width of the inputend of the chamber 10, serve to introduce a sheet of fluid that travelsthroughout the chamber 10 in a substantially linear direction.

The introduced fluid carries the particulate matter through the chamber10. This fluid may be, for example an eluate, such as a cell-freesuspension having a mixture of dextrose and sucrose, tissue culturemedium, non-ionic or zwitterionic solutions, or other suspension mediumsor non-biological oils, solvents such as phenol, alcohol, CCl₄, ethyleneglycol, or others known in the art. Following transit through thechamber 10, fluid leaves at the opposite end through an exit port. Thisexit end of the chamber 10 may include, for example, one or more exitports 30, which may be arranged in one or more arrays of ports as shownin FIG. 2A. In the absence of an applied field, that is, when noelectrical signal is applied to the electrode elements 5, particles movethrough the chamber 10 under the influence of fluid flow. This fluid canbe controlled to flow at different speeds. Further, based on thegeometrical design of the chamber 10, the fluid may exhibit, forexample, a laminar flow, in which the speed of the flow is fastesttowards the center of the chamber 10. That is, the hydrodynamic flowprofile is along a horizontal plane. Simultaneous to the influence ofthe fluid flow, the particles undergo sedimentation due to gravitationalforces on the particles, so that they exit the chamber 10 atcharacteristic heights determined by their sedimentation rates.

When electrical signals are applied, however, the particles experiencecDEP forces that cause them to move to characteristic distances, knownas an equilibrium position, from the side walls of the chamber 10 wherethe electrode arrays 5 are arranged. In this embodiment, differentelectrical signals (frequency or magnitude or both) are applied toelectrode elements 5 on each of the side walls. Different particlesequilibrate at different characteristic distances from the side walls ofthe chamber, based on the synergism of the differing electrical signals,which create an inhomogeneous electric field, causing DEP forces on theparticles. These different signals cause different particles toequilibrate at different characteristic distances from the side walls ofthe chamber, based on the synergism of the DEP forces caused by theelectric field created by the differing signals.

Such particle equilibration depends on the dielectric and conductiveproperties of the particles, the magnitude and frequency of theelectrical fields applied to the electrodes on the facing chamber walls,and the fluid density, viscosity and flow rate as shown in FIG. 2B.Matter introduced into chamber 10 travels at different positions fromelectrode arrays 5. The velocities of the different particles within thefluid are controlled by the velocity profile of the fluid. Because fluidflowing through a thin chamber sets up a velocity profile, particlesthat have equilibrated at different distances from the chamber wallswill be carried at different velocities and therefore take varyingamounts of time to traverse the chamber. The fluid flows at a maximumrate towards the center of the chamber, with this rate proportionatelydiminishing as distance to the side walls decreases. The fluid flow ratemay be between about 0.1 μl/min. and about 1000 μl/min., and morepreferably between about 1 μl/min. and about 100 μl/min. The skilledartisan will recognize, however that variations in the dimensions of theapparatus will affect the fluid flow rate, and that the indicated flowrates are illustrative for the dimensions of the present apparatus.

The distance that particles sediment during their passage across thechamber will depend upon their transit time, as gravity forces act onthe particles during their transit through the chamber. Consequentlydifferent particles will sediment to different depths based upon theparticle's transit time through the chamber 10. Particle sedimentationalso depends on particle characteristics, such as size, mass, andvolume, for example. Therefore, the time required for particles totravel across the entire length of the chamber is controlled by thefluid flow profile. The placement of particles within the fluid flowprofile is in turn determined by the synergism of the differingelectrical signals. Discrimination may be accomplished either in "batchmode" or in "continuous mode." In batch mode, an aliquot of particles isinjected and collected with respect to the time of transit for theparticles and the height of exit at the outlet ports 30. In continuousmode, a constant stream of particles is injected into the inlet port,and particles emerging at different heights are continuously collected.

In an apparatus according to the present invention, it is possible tovary the carrier fluid characteristics at different heights with respectnot only to flow rate but also to fluid density, dielectricpermittivity, pH and conductivity. In this way additional particlecharacteristics may be exploited for particular separation applications.

In the general case, the device may be oriented at any angle to takeadvantage of discriminating aspects of the horizontal and vertical casesdescribed above. In this generalized situation the particle density,sedimentation rate and dielectric properties, together with allcomponents of the cDEP force are utilized. Separation in continuous orbatch mode is possible. Different embodiments of an apparatus accordingto the present invention may have additional components connected to theoutlet ports 30. For example, particles emerging from the exit ports 30of the apparatus of the present invention may be collected by one ormore fraction collectors, or the like. Additionally, the matter may bemeasured by one or more measuring or characterizing structures such as acytometer, for example. Furthermore, when necessary, particles may betransferred to collection wells containing appropriate solutions ormedia, such as neutral salt buffers, tissue culture media, sucrosesolutions, lysing buffers, solvents, fixatives and the like to trapcells exiting the chamber.

An alternate method of operation of this second embodiment utilizes thetwDEP force, which acts in a substantially vertical direction from thecombined fields produced by the electrode arrays 5. Different electricalsignals (frequency, magnitude, and phase, or a combination thereof) maybe applied to the facing electrodes 5 from the signal generator so thatparticles experience different cDEP and/or twDEP forces. The cDEP forcesmay act to displace matter to or away from the electrode plane andthereby equilibrate at an equilibrium position in the fluid flow stream.Matter also sediments as it travels through chamber 10. The distancethat matter sediments as it travels depends on the time required totraverse chamber 10 and the sedimentation rate of the matter.

In this alternate method, the distance of matter sedimentation is alsoaffected by the twDEP force. Specifically, the twDEP force acts in asubstantially vertical direction when electrode arrays 5 are located onthe side walls of chamber 10 and arranged substantially parallel to thefluid flow. Therefore the twDEP force component adds to or opposes thesedimentation forces acting on the matter. Thus the net verticaldisplacement which the matter experiences as it traverses chamber 10 ismodified.

Discrimination in this alternate method may be accomplished in acontinuous mode, whereby a constant stream of matter is injected intothe inlet ports, and matter emerging at differing vertical displacementsis continuously collected at the outlet ports. It may be possible tooperate this embodiment in the pulse mode. To further achieve thebenefits of discrimination, it may be possible to vary the carrier fluidcharacteristics at different vertical displacements by varying flowrate, fluid density, viscosity, dielectric permittivity, pH andconductivity of the carrier fluid. In this way, additional mattercharacteristics may be exploited for particular discriminationapplications.

Methods of Operation

The following descriptions detail construction of an apparatus andmethods of operation according to the present invention.

In one embodiment, an apparatus according to the present invention wasconstructed using two glass slides (1"×1.5", for example) as chamberwalls. These walls may be spaced by Teflon spacers; however othermethods of separating chamber walls, such as glue, polymer gaskets, ormechanical precision clamps may be used, for example. The distance ofseparation between walls may be between about 0.1 microns and about 1000microns, and more preferably between about 10 microns and about 200microns. In studies using the present apparatus, the distance ofseparation was 127 microns. One wall of the chamber supported amicroelectrode array consisting of about 20 micron wide parallelelectrode elements spaced about 20 microns apart. The electrode elementsmay run along the entire length of the chamber from the input port tothe output port. It is understood that the length, width, thickness andspacing of electrode arrays may be altered to create electric fields ofdiffering intensities and different inhomogeneity. It is also to beunderstood that an array of electrodes may be used with the presentinvention, or a single electrode element may be sufficient for certainapplications, if combined with a ground plane. Further, it is to beunderstood that the electrode array may not be parallel, and othergeometric configurations, such as serially arranged electrodes, linear,polynomial, interleaved, three-dimensional and the like may be utilized.

In an exemplary embodiment, alternate electrode elements may beconnected to electrode buses along the two opposite long edges of thechamber wall. These electrode buses are connected to an electricalsignal generator, which may be, for example, a function generator. Othersuitable signal generators may include, for example, oscillators, pulsegenerators, digital output cards, klystrons, RF sources, masers, or thelike. The electrode array may be fabricated using standardmicrolithography techniques, as are known in the art. For example, theelectrode array may be fabricated by ion beam lithography, ion beametching, laser ablation, printing, or electrodeposition. The electrodearray of the exemplary embodiment described herein consisted of 100 nmgold over a seed layer of 10 nm chromium. It is understood that thepresent invention contemplates using electrical signals in the range ofabout 0 to about 15 V and about 0.1 kHz to about 180 MHz, and morepreferably between about 10 kHz and about 10 MHz. In studies which aredescribed below, the signals were provided by a HP 8116A functiongenerator. The present invention may utilize a fluid flow of about 0.1μl/min. to about 500 μl/min., and more preferably between about 1 μl/minand about 50 μl/min. In studies described below, fluid flow in the rangeof about 1-100 μl/min, was provided by a digital syringe pump.

Field Flow Fractionation

Cell mixtures in the studies discussed below consisted of blood cells(collected by venipuncture from healthy volunteers and diluted with 90parts Ca²⁺ /Mg²⁺ -free PBS containing 5 mM hemisodium EDTA) mixed in aratio of 3:2 with HL-60 leukemia cells that had been cultured understandard conditions and harvested by centrifugation. Cell mixtures werewashed twice in isotonic (8.5%) sucrose containing 3 mg/ml dextrose andresuspended at a final concentration of 2×10⁷ malignant cells and 3×10⁷normal blood cells per ml in this same medium. The suspensionconductivity was adjusted to 10 mS/m by addition of hemisodium EDTA to afinal concentration of approximately 0.7 mM. It is contemplated by thepresent invention that other methods of obtaining and preparing samplesare acceptable. Further, different ratios of the mixture may be used.For example, cell mixtures may be washed twice in an isotonic solutionof 8.5% sucrose and 0.3% dextrose, resuspended at a final concentrationof 1×10⁷ malignant cells and 3×10⁷ normal blood cells per ml in thissame medium, and adjusted to a conductivity of 10 mS/m with a finalconcentration of ˜0.7 mM hemisodium EDTA.

FIG. 3A shows the results of field flow fractionation on a sample ofHL-60 cells (ATCC) cultured in a medium of RPMI 1640 10% FBS 22 mM HEPESin an apparatus as described above. The fractionation occurred at a flowrate of 200 μl/min. As shown in FIG. 3A, a sharp rise in HL-60 cellsexiting the apparatus occurs at approximately 10 minutes after the flowof cells began. After this rise, the cell count rapidly tapers to alower level which continues for approximately 50 minutes. FIG. 3Bsimilarly shows the results of field flow fractionation of HL-60 cellsat a flow rate of 100 μl/min. As shown in FIG. 3B, a sharp rise in HL-60cells exiting the chamber occurs at approximately 30 minutes after theflow of cells began. Again, after this rise, the cell count rapidlytapers to a lower level which continues for approximately 30 minutes.

FIG. 3C shows the results of field flow fractionation on a mixture ofHL-60 and human whole blood in a medium of 8.5% sucrose and 3 mg/mldextrose adjusted to mS/ml, in an apparatus as described above. Thefractionation occurred at a flow rate of 100 μl/min. As shown in FIG.3C, a sharp rise in the HL-60 cells exiting the chamber occurred atapproximately 20 minutes after the flow began. Thereafter, a second risein the number of cells exiting occurred at approximately 60 minutes,which correlated to the exit of the human blood cells. However, it isnoted that cells continue to exit before and after the peaks. Thus,separation by field flow fractionation is not capable of a completeseparation. Therefore, FIGS. 3A, 3B, and 3C demonstrate that althoughfield flow fractionation may discriminate and separate some particles ofdifferent characteristics, there is needed greater discriminationcapabilities.

Three types of studies utilizing the apparatus of the present inventionwere performed that caused cDEP forces on the particulate matter:

(1) Levitation of Cells Caused by cDEP Force

The levitation of DS-19 murine erythroleukemia cells (M. Rifkind)supported in 8.5% sucrose +0.3% dextrose solution having a conductivityof 56 mS/m was investigated as a function of the frequency and voltageof signals applied to the electrode array in the absence of fluid flow.It is to be understood that various solutions having conductivities inthe range of about 10 μS/m to about 2 S/m, such as tissue culture mediumor the like, may be used. Further, it is possible to utilize acollection of cells only. Other solutions may be practicable so long asthe conductivity of the solution is either much more conductive or muchless conductive than the cell interiors.

The results of this study are shown in FIG. 4A and FIG. 4B. In thefrequency range 1 kHz-40 kHz, DS19 cells were levitated to about 20microns at an applied voltage of 4 V peak to peak (p--p), as shown inFIG. 4A. Above 40 kHz, the levitation height dropped rapidly, and whenthe frequency reached 140 kHz and above, cells were no longer levitatedbut were instead attracted to electrode edges by positive cDEP.

At an applied frequency of 50 kHz, levitation of DS19 cells occurredwhen the applied voltage was above about 0.5 V p--p, as shown in FIG.4B. Above this threshold, the cells levitated and the height oflevitation increased with increasing voltage. This behavior wasconsistent with that predicted by cDEP theory, the dielectric propertiesof the cells as measured using the technique of electrorotation, and thedensity of the cells and their supporting medium.

(2) Combined FFF/cDEP

A second study using the apparatus discussed above involved the velocityof HL-60 human promyclocytic leukemia cells supported in 8.5% sucrose+0.3% dextrose solution having a conductivity of about 10 mS/m with anestablished fluid flow in the chamber, as a function of the frequency ofthe voltage signals applied to the electrode array. When no voltagesignal was applied, the cell velocity was about 10 microns per second asthey were transported under the influence of an applied fluid flow rateof 10 μl/min. The fluid flow may be either the solution including thecells to be tested, or it may be another fluid, or the same fluidwithout the cells. Additionally the solution may be ramped over time toalter, for example, the pH, or conductivity of the solution.

As shown in FIG. 5A, addressing the electrodes with voltage signalsaffected the height at which the cells traveled above the chamber bottomwall and thereby altered their position and velocity in the laminarflow. Below 10 kHz, cell velocity increased to about 50 microns persecond with an applied voltage of 3 V p--p. As the frequency wasincreased in the range of about 10 kHz to about 25 kHz, the cellvelocity gradually fell as the levitation height was reduced. Above 30kHz, these cells were attracted to the electrode and thus they ceasedmoving. This response with increasing frequency agreed with the behaviorexpected from the measured electrical properties of the cells.

As shown in FIGS. 5B and 5C, similar results were obtained for studiesusing other cells having different cell properties. Specifically, FIG.5B shows the results for MDA 468 cells (kindly supplied by Janet Price)in a solution of 8.5% sucrose 0.3% dextrose conductivity at 10 ms/m at aflow rate of 40 μl/min at 3 V p--p. FIG. 5C shows the results forMDA-435 cells (kindly supplied by Janet Price) in the same solution at aflow rate of 40 μl/min at 3 V p--p. FIG. 5D shows the results forMDA-435 cells at a flow rate of 40 μl/min at a frequency of 31.6 kHz. Asnoted in FIG. 5D, the velocity of cells increases approximately linearlywith voltage.

(3) cDEP/FFF on Mixture of HL-60 and Human Blood Cells

The chambers of the apparatus were preloaded with a mixture of HL-60 andhuman blood cells in the ratio 1:10 at a total concentration of 5×10⁷cells/ml. The cells were supported in 8.5% sucrose +0.3% dextrosesolution having a conductivity of 10 mS/m. A voltage of 3 V p--p at 40kHz was applied to the electrodes and fluid flow at the rate of 10μl/min was started. All of the HL-60 cells were trapped at the edges ofthe electrode elements, while the human blood cells (mainlyerythrocytes) were levitated and were transported by the fluid. Byadjusting the frequency in the range of 8-15 kHz, HL-60 cells were alsoreleased and their rate of transport controlled relative to theerythrocytes. When HL-60 cells were levitated to heights above or belowthe erythrocytes, they moved correspondingly more quickly or more slowlythan these blood cells depending on their position in the field flow.

The following is an additional study performed according to the presentinvention. Fluids were injected and removed through slots at each end ofthe chamber. The outlet port was furnished with a well to trap cellsexiting the chamber. Prior to performing studies, the chamber was soakedfor 5 minutes with 20% (w/v) bovine serum albumin solution to render theglass surfaces less adherent to cells. Alternately the glass surfacesmay be air blown, or washed and treated with silane. Dielectrophoreticforces were generated by connecting alternate electrodes to sinusoidalvoltages of fixed or swept frequencies, and were monitored using anoscilloscope. Forces to remove cells from the separation chamber wereprovided by laminar flow of an eluate buffer, controlled by two digitalsyringe pumps connected in push-pull configuration between the inlet andoutlet ports of the chamber. A bubble-free path of fluid was maintainedbetween the pumps at all times.

Following injection of approximately 30 μl of the cell mixture (about1.2×10⁶ cells) to half fill the chamber, a 200 kHz signal of 5 Vpeak-peak was applied to the electrode array for 30 sec to collect allcells by positive DEP at the high-field regions of the electrode tips.It is not required, however, to only half-fill the chamber, and a largerchamber may allow for better discrimination. Flow of eluate (consistingof cell-free suspension buffer, which may also be a mixture of 8.5%sucrose plus 3 mg/ml dextrose having a conductivity of 10 mS/m), wasthen started at 5 μl/min. This flow may be accomplished under thecontrol of two digital syringe pumps operating in a push-pullconfiguration between the inlet and outlet ports of the chamber.Alternately, the flow may be controlled by a peristaltic pump, gravityflow, blood pressure, or the like. The frequency of the applied electricsignal was lowered until the tumor cells were selectively retained whilethe blood cells were eluted and trapped in the collection well. After 20minutes, cells were removed from the well by cross-flow between twoadditional syringe ports without disturbing the tumor cells still on theelectrodes. The voltage was then turned off to release the cells held byDEP and these were eluted and collected separately.

In a further embodiment, an apparatus according to the present inventionwas constructed using two glass slides (1"×3", for example) as chamberwalls. These walls were spaced apart by TEFLON spacers with a distanceof separation of approximately 50 microns. The apparatus may be arrangedso that the glass slides comprise the side walls, and the spacers thetop and bottom walls. Alternately, the apparatus may be arranged so thatthe glass slides comprise the top and bottom walls, and the spacerscomprise the side walls. In this exemplary embodiment, the top andbottom walls supported microelectrode arrays consisting of about 20micron wide parallel electrode elements spaced about 20 microns apart.The electrode arrays may be configured so that the electrode elements onone wall align with the spaces between electrode elements on the otherelectrode array. The electrode arrays may be further configured so thatthe electrode elements are arranged along the long walls of the chamberside walls so that the electrodes are aligned substantially parallel tothe fluid flow. It is understood that the length, width, thickness andspacing of electrode arrays may be altered to create electric fields ofdiffering intensities and different inhomogeneity, and travelingspatially as a function of varying phase. However, it is also to beunderstood that the electrode arrays need not be parallel, and othergeometric configurations, such as serially arranged electrodes, linear,polynomial, interleaved, three-dimensional and the like may be utilized.

In an exemplary embodiment, alternate electrode elements may beconnected to electrode buses along the short ends of the chamber sidewalls. The electrode buses on one chamber side wall may receiveelectrical signals having 0° and 180° relative phase, and the buses onthe other chamber side wall may receive signals having 90° and 270°relative phase. It is understood that alternate electrode elements mayreceive signals having relative phases of differing values. Theseelectrode buses are connected to an electrical signal generator, whichmay be, for example, a digital synthesizer. Other suitable signalgenerators may include, but not be limited to, oscillators, functiongenerators, pulse generators, digital output cards, klystrons, RFsources, masers, or the like. The electrode array of the embodimentdescribed herein consisted of 100 nm gold over a seed layer of 10 nmchromium fabricated by standard microlithographic techniques. It isunderstood that the present invention contemplates using electricalsignals in the range of about 0 to about 15 V and about 0.1 kHz to about180 MHz, and more preferably between about 10 kHz and about 10 MHz. Inthe study described below utilizing this apparatus, the signals wereprovided by a custom-built digital synthesizer. The present inventionmay utilize a fluid flow of about 0.1 μl/min. to about 500 μl/min., andmore preferably between about 1 μl/min and about 50 μl/min. In studiesdescribed below, fluid flow in the range of about 1-100 μl/min, wasprovided by a digital syringe pump.

(1) Levitation of Cells Caused by gDEP Force

The levitation of DS-19 murine erythroleukemia cells (M. Rifkind)supported in 8.5% sucrose +0.3% dextrose solution having a conductivityof 10 mS/m was investigated as a function of the frequency of signalsapplied to the electrode array both in the absence of fluid flow andwith a fluid-flow. It is to be understood that various solutions havingconductivities in the range of about 10 μS/m to about 2 S/m, such astissue culture medium or the like, may be used. Further, it is possibleto utilize a collection of cells only. Other solutions may bepracticable so long as the conductivity of the solution is either muchmore conductive or much less conductive than the cell interiors.

The DS19 cell mixed in the suspension medium was introduced into thechamber at a flow rate of 10 μl/min. As the frequency of the electricalsignals was gradually increased from approximately 1 kHz toapproximately 15 MHz at 3 volts peak-to-peak, three kinetic effects wereobserved:

(i) 1 to 30 kHz. Cells were levitated above the electrode plane by cDEPforces (typically by about 18 μm, depending on frequency) and movedcounter to the applied travelling wave by twDEP forces at rates thatincreased with increasing frequency to approximately 40 microns/sec.Fast-moving cells overtook slow ones without forming pearl-chainscommonly observed when cDEP alone is used for cell manipulation. In thepresence of fluid flow applied at right angles to the twDEP forces at aflow rate of 10 μl/min., individual cells were deflected to differentdegrees depending on their electrical characteristics. Furthermore, thevelocity of cells due to the fluid flow depended on their distances fromthe electrode plane in accordance with cDEP/FFF principles.

(ii) 30 to 50 kHz. The cDEP force acting on the cells was very small andcells remained close to the electrodes at the bottom of the chamber.Some cells were deflected in the same direction as the travelling waveby the twDEP forces, other cells were barely deflected, and some cellswere deflected in the opposite direction to the twDEP waves. In thepresence of fluid flow at a flow rate of 10 μl/min., maximal differencesin cell velocity in the fluid stream occurred due to cDEP/FFFinfluences, and twDEP forces resulted in different degrees of deflectionacross the fluid stream for different cells.

(iii) 50 kHz to 15 MHz. Cells were mostly attracted to electrode edgesand immobilized under the influence of strongly positive cDEP forces.

These findings agreed with the predictions of generalizeddielectrophoresis. They show further that the combined influence of cDEPand twDEP make possible a new type of cell sorting that depends on boththe real and imaginary components of the cell electrical polarizabilityas well as the suspending medium dielectric and conductive properties.

It is contemplated that the apparatus and methods according to thepresent invention may be used for cell or particle characterization, asa diagnostic tool to identify, for example, cancer cells or other cellsthat are desired or of interest to the clinician, and as a therapeutictool to purge a patient sample of undesired cells or other particle.

For example, the methods according to the present invention may be usedto characterize the physical properties of an unknown particulatematter. A sample including an unknown biological or organic or mineralsample may be input into the chamber and separated according to theprocedures set forth above. Following separation and removal ofextraneous particles, the unknown particle may be collected at an outputport of the chamber. The particle can then be analyzed using standardparticle characterization techniques known in the art, such as thoseused in diagnostic microbiology and in histology, for example, electronmicroscopy. After determining characteristics that are unique to aparticle, an investigator may then compare these characteristics to theknown characteristics of a particle. Therefore, the researcher maydetermine whether the unknown particle is the same as a known particle,or whether it has similar properties.

In addition, the invention contemplates the characterization of knownparticles. which may then be used as a reference tool for determiningunknown particles based on similar trapping frequencies, voltages, flowrates, and other parameters set forth above. The sample may beintroduced into the chamber of the present invention and then besubjected to the separation methods detailed above. By performing theseseparation techniques, the trapping frequency and release frequency ofthe particle can be determined. These values are then useful incomparing similar parameters of an unknown sample to this known sample.Certain clinical applications requiring separation of a known particlefrom an unknown particle would require such values to complete themethods of separation.

A clinical application of the present invention would be to use thepresent apparatus and methods as a diagnostic tool to screen unknownsamples for the presence or absence of various cell types. First, as setforth previously, a patient's sample may be placed in the apparatus, andvarious cell types may be separated based on previously determinedparameters or characteristics. These cells may include cancer cells, orcells infected with bacteria, viruses, protozoans, or parasites,bacteria, viruses protozoans, or they may include cells that aredeficient in certain enzymes or cell organelles, altered biopsies,plaques and scrape tests including Pap smears and so forth. Thus, it iswell within the scope of the invention to separate all types ofparticles that have differential sedimentation rates in a fluid stream,based on size, density, dielectric strength, and conductivity, forexample. Therefore, the present invention may be used to diagnose thepresence of a condition, for example, a cancer, or other cellulardisorder.

Another clinical application would be to use the apparatus and methodsof the present invention to separate unwanted cells, such as cancerouscells, from a cell population including wanted or normal cells. Forexample, once a cancer has been detected, for instance in bone marrow, apatient's bone marrow may be input into an apparatus according to thepresent invention to separate the cancer cells, or preneoplastic cells,from normal cells. These normal cells may then be collected at theoutput of the chamber and returned to the patient, while the unwantedcancer cells may be later collected at the output of the chamber andcharacterized, utilized in further studies, or discarded. In thismanner, unwanted cells are purged from a normal cell population, whileat the same time a particular cell type is enriched, such as tumorcells, normal cells, progenitor cells, etc.

The apparatus and methods disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the apparatus and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to theapparatus, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. All substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An apparatus for the discrimination of matterutilizing dielectrophoresis and field flow fractionation, comprising:a)a chamber having an inlet port and a plurality of outlet ports, saidchamber having an interior surface and an exterior surface, said chamberfurther having a substantially greater width than thickness which causesfluid traveling through said chamber to travel at different velocitiesaccording to a velocity profile; b) a plurality of electrode elementsadapted along a portion of said chamber, wherein the plurality ofelectrode elements comprise an electrode array; and c) wherein saidplurality of electrode elements energized at different phases byelectrical signals provided by an electrical signal generator creates atraveling electric field, thereby causing a dielectrophoretic force onsaid matter having components normal to the direction of said fluidtraveling through said chamber, said dielectrophoretic force displacessaid matter to varying distances in said velocity profile and in a planeparallel with respect to said plurality of electrode elements such thatsaid matter is not collected on said plurality of electrode elements. 2.The apparatus according to claim 1, wherein said plurality of outletports are vertically lower than said inlet port, thereby permittingdiscrimination of said matter by sedimentation.
 3. The apparatusaccording to claim 1, wherein said dielectrophoretic force acts solelyin a direction normal to said fluid traveling through said chamber. 4.The apparatus according to claim 1, wherein each of said plurality ofelectrode elements is individually connected to one of a plurality ofelectrical conductors electrically connected to said electrical signalgenerator.
 5. The apparatus according to claim 1, wherein said energizedplurality of electrode elements further creates a spatiallyinhomogeneous electric field.
 6. The apparatus according to claim 1,wherein said positions of said matter within said fluid causes saidmatter to travel through said chamber at velocities according to saidvelocity profile.
 7. The apparatus according to claim 1, wherein saidmatter exits from said plurality of outlet ports at time intervalsproportionate to its position within said velocity profile.
 8. Theapparatus according to claim 1, wherein said matter exits from saidplurality of outlet ports at positions laterally displaced from saidinlet port.
 9. The apparatus according to claim 1 wherein the velocityprofile is a hydrodynamic fluid profile.
 10. The apparatus according toclaim 7, wherein the hydrodynamic fluid profile is a parabolic flowprofile.
 11. The apparatus according to claim 1, wherein saiddielectrophoresis comprises conventional dielectrophoresis.
 12. Theapparatus according to claim 1, wherein said dielectrophoresis comprisestraveling wave dielectrophoresis.
 13. The apparatus according to claim1, wherein said dielectrophoresis comprises generalizeddielectrophoresis.
 14. The apparatus according to claim 1, wherein saidchamber comprises a tube.
 15. The apparatus according to claim 12,wherein said plurality of electrode elements is adapted along theinterior surface of said tube.
 16. The apparatus according to claim 12,wherein said plurality of electrode elements is adapted along theexterior surface of said tube.
 17. The apparatus according to claim 1,wherein the chamber includes a top wall, a bottom wall, and two sidewalls.
 18. The apparatus according to claim 1, wherein said mattercomprises particulate matter.
 19. The apparatus according to claim 16,wherein the particulate matter is selected from the group consisting ofa cell, cell aggregate, cell organelle, nucleic acid, bacteria,protozoan, and virus.
 20. The apparatus according to claim 1, whereinsaid matter comprises solubilized matter.
 21. The apparatus according toclaim 18, wherein the solubilized matter is selected from the groupconsisting of a molecule, molecular aggregate, and molecule mixture. 22.The apparatus according to claim 19, wherein the molecule or molecularaggregate is selected from the group consisting of a protein and aprotein mixture.
 23. The apparatus according to claim 1, wherein theplurality of electrode elements are adapted substantially longitudinallyalong a portion of said chamber.
 24. The apparatus according to claim 1,wherein the plurality of electrode elements are adapted substantiallylatitudinally along a portion of said chamber.
 25. The apparatusaccording to claim 1, wherein the plurality of electrode elements areadapted along the interior surface of said chamber.
 26. The apparatusaccording to claim 1, wherein the plurality of electrode elements areadapted along the exterior surface of said chamber.
 27. The apparatusaccording to claim 1, wherein a ratio of electrode element width toelectrode element spacing is modified to change levitation heights ofsaid matter.
 28. The apparatus of claim 1, wherein said plurality ofelectrode elements are configured on a plane substantially normal to aflow of said fluid travelling through said chamber.
 29. The apparatus ofclaim 1, wherein said plurality of electrode elements are configured ona plane substantially parallel to a flow of said fluid travellingthrough said chamber.
 30. The apparatus of claim 1, wherein saidplurality of outlet ports are connected to a fraction collector.
 31. Theapparatus of claim 1, wherein said plurality of outlet ports areconnected to collection wells.
 32. The apparatus according to claim 1,wherein the electrode array is configured on a top wall of said chamber.33. The apparatus according to claim 1, wherein the electrode array isconfigured on a bottom wall of said chamber.
 34. The apparatus accordingto claim 1, wherein a plurality of said electrode arrays are adapted onopposing surfaces of said chamber.
 35. The apparatus according to claim1, wherein a plurality of said electrode arrays are adapted on opposingsurfaces of said chamber such that each of said electrode elements isnot directly opposed by another said electrode element on said opposingsurface.
 36. The apparatus according to claim 1, wherein said velocityprofile is in a substantially horizontal plane.
 37. The apparatusaccording to claim 1, wherein said electrical signal generator iscapable of varying magnitude, or frequency, and phase of said electricalsignals.
 38. The apparatus according to claim 1, wherein more than twoelectrical signals having different phases are provided by saidelectrical signal generator.
 39. An apparatus for the discrimination ofmatter, comprising:a) a chamber having an inlet port and a plurality ofoutlet ports, said chamber having an interior surface and an exteriorsurface, a top wall, a bottom wall and two side walls, said chamberhaving a substantially greater width than thickness which causes fluidtraveling through said chamber to travel at different velocitiesaccording to a velocity profile; b) an electrode array adapted along aportion of each of at least two said walls in opposing relation, each ofsaid electrode arrays electrically connected to electrical conductors;c) a duct entering the chamber, said duct capable of introducing saidfluid to carry said matter from an inlet port of said chamber to aplurality of outlet ports of said chamber; d) an electrical signalgenerator electrically connected to said electrical conductors; and e)wherein said electrode arrays energized at different phases byelectrical signals provided by said electrical signal generator create atraveling electric field in said chamber, thereby causing adielectrophoretic force on said matter to displace said matter tovarying distances in said velocity profile with respect to saidelectrode arrays, and to displace said matter parallel to said electrodearrays such that said matter is not collected on said electrode arrays.40. An apparatus according to claim 39, wherein said inlet port isadapted in close relation to said top wall.